Epithelial cells along the mucosal surface provide the front line of host defense against pathogen infection in the gastrointestinal (Gl) tract. Because of the importance of Toll-like receptor (TLR) signaling in the initiation and regulation of Gl mucosal immunity, the overall objective of this application is to better understand the molecular mechanisms by which TLR signaling coordinates Gl epithelial antimicrobial defense. Our preliminary studies demonstrate that microbial challenge stimulates exosome release from the apical side of cultured Gl epithelial monolayers in a TLR4-dependent manner. Released exosomes shuttle a variety of antimicrobial peptides and display antimicrobial activity ex vivo. Moreover, activation of TLR4/NF-?B signaling causes alterations in expression of microRNAs (miRNAs), small non-coding miRNAs that regulate gene expression at the posttranscriptional level. Selected TLR4-responsive miRNAs may target effector molecules that regulate the exocytotic process and, thus, are potentially involved in TLR4-mediated exosome release. Based on these exciting novel preliminary data, we propose to test the hypothesis that the release of exosomes from epithelial cells is regulated by TLR signaling with the involvement of miRNA- mediated gene regulation at the posttranscriptional level, and that it contributes to TLR-mediated Gl epithelial antimicrobial defense. We will use in vitro and in vivo infection models and complementary biochemical, molecular, and morphologic approaches to test four interrelated Specific Aims: i) The TLR signaling pathway regulates release of apical exosomes from epithelial cells in response to microbial challenge; ii) TLR signaling stimulates exosome release from epithelial cells through the IKK/SNAP-23- associated exocytotic process with the involvement of miRNA-mediated posttranscriptional regulation; iii) TLR signaling regulates exosomal shuttling of antimicrobial peptides; and iv) epithelial exosomes contribute to TLR-mediated epithelial antimicrobial defense. The proposal is conceptually innovative as it tests new concepts regarding TLR-mediated mucosal antimicrobial defense. The information obtained from this study should provide a rational basis for the design and implementation of new therapeutic strategies.